KU-0063794 is a potent and highly specific dual-mTOR inhibitor of mTORC1 and mTORC2 with IC50 of ~10 nM in cell-free assays; no effect on PI3Ks.
Effects of PI3K and mTOR inhibitors on IGF1R and AKT signaling in RMS cells. Rh41 cells were treated with 0.3 uM PI3K inhibitor BKM120, 0.3 uM mTOR inhibitor KU0063794 for the indicated time. Lysates were made and analyzed for p-AKT, S6RP and IGF1R.
Oncogene 2013 10.1038/onc.2013.509. KU-0063794 purchased from Selleck.
mTORC2 is required for mechanical activation of Akt. A, immunoblots of mdMSC subjected to strain for 45 min following treatment with the mTOR inhibitor KU0063794 (2uM). B, densitometric analysis of phosphorylated GSK3 (Ser-9) normalized to total GSK3. D, immunoblots of mdMSC treated with KU0063794 and then stimulated with insulin (50 nM) for 60 min.
J Biol Chem 2011 286, 39450-6. KU-0063794 purchased from Selleck.
mTORC2-mediated stretch-induced SGK-1 phosphorylation. SMCs infected with Ad-SGK-1 were pretreated with PPP (10 μmol/L), rapamycin (100 nmol/L),or Ku-0063794 (10 μmol/L) for 30 minutes and then were subjected to stretch or IGF-1 (10 ng/mL) for 30 minutes.
Circ Res 2010 107, 1265-1274. KU-0063794 purchased from Selleck.
For MTT assays, cells (2,000 ~ 5,000 cells/well) were subcultured into 96-well plates according to their growth properties. Cell proliferation was assayed at 72 hr after treatment of KU-0063794 by adding 20 μl of 5 mg/ml 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution per 100 μl of growth medium. After incubating for 3-4 h at 37°C, the media were removed and 150 µl/well of MTT solvent (either absolute DMSO or isopropanol containing 4 μM HCl and 0.1% Nonidet-40) was added to dissolve the formazan. The absorbance of each well was measured by ELx808 (BioTek, Winooski, VT) or Wallac Victor2 (Perkin-Elmer Life Sciences, Boston, MA) Microplate Reader. Viable cells are presented as percent of control, vehicle-treated cells.
Dr. Yong-Weon Yi from Georgetown University Medical Center. KU-0063794 purchased from Selleck.
|製品説明||KU-0063794 is a potent and highly specific dual-mTOR inhibitor of mTORC1 and mTORC2 with IC50 of ~10 nM in cell-free assays; no effect on PI3Ks.|
Compared with the mTOR inhibitor PP242, KU-0063794 exhibits higher specificity for mTOR, as being inactive against PI3Ks or 76 other kinases. In HEK-293 cells, KU-0063794 at 30 nM is sufficient to rapidly ablate S6K1 activity by blocking the phosphorylation of the hydrophobic motif (Thr389) and subsequently the phosphorylation of the T-loop residue (Thr229). In case of IGF1 stimulation of serum-starved HEK-293 cells, 300 nM of KU-0063794 is needed to inhibit the S6K1 activity by ~90%. KU-0063794 at 100-300 nM also completely inhibits the amino-acid-induced phosphorylation of S6K1 and S6 protein. Similar to S6K1, KU-0063794 inhibits the phosphorylation of mTORC1 at Ser2448 and mTORC2 at Ser2481 in a dose-dependent and time-dependent manner. In the presence of serum or following IGF1 stimulation, KU-0063794 induces a dose-dependent inhibition of the activity and phosphorylation of Akt at Ser473 and unexpected Thr308 as well as the phosphorylation of the Akt substrates PRAS40 at Thr246, GSK3α/GSK3β at Ser21/Ser9 and Foxo-1/3a at Thr24/Thr32. KU-0063794 but not rapamycin inhibits SGK1 activity and Ser422 phosphorylation as well as its physiological substrate NDGR1 in a dose-dependent manner, to the same extent as S6K1 and Akt phosphorylation, whereas KU-0063794 dose not inhibit phorbol ester induced ERK or RSK phosphorylation and RSK activation. Compared with rapamycin, KU-0063794 exhibits more significant potency to induce the complete dephosphorylation of 4E-BP1 at Thr37, Thr46 and Ser65. KU-0063794 inhibits cell growth of both wild-type and mLST8-deficient MEFs and induces a G1 cell cycle arrest, more significantly than rapamycin. 
|体内試験||Ku0063794 inhibits tumor growth and mTOR signaling in a preclinical renal cell carcinoma model. However, Ku0063794 was not more effective than temsirolimus in the animal study. A possible explanation for lack of greater activity in vivo for Ku0063794 is that temsirolimus has important effects on the tumor microenvironment. Temsirolimus decreased angiogenesis in the xenograft tumors while Ku0063794 did not. Temsirolimus treated tumors expressed less VEGF and PDGF than Ku0063794 treated tumors, thus stimulating less angiogenesis.|
mTOR complexes kinase assays:HEK-293 cells are freshly lysed in Hepes lysis buffer. Lysate (1-4 mg) is pre-cleared by incubating with 5-20 μL of Protein G-Sepharose conjugated to pre-immune IgG. The lysate extracts are then incubated with 5-20 μL of Protein G-Sepharose conjugated to 5-20 μg of either anti-Rictor or anti-Raptor antibody, or pre-immune IgG. All antibodies are covalently conjugated to Protein G-Sepharose. Immunoprecipitations are carried out for 1 hour at 4 °C on a vibrating platform. The immunoprecipitates are washed four times with Hepes lysis buffer, followed by two washes with Hepes kinase buffer. For Raptor immunoprecipitates used for phosphorylating S6K1, for the initial two wash steps the buffer includes 0.5 M NaCl to ensure optimal kinase activity. GST-Akt1 is isolated from serum-deprived HEK-293 cells incubated with PI-103 (1 μM for 1 hour). GST-S6K1 is purified from serum-deprived HEK-293 cells incubated with rapamycin (0.1 μM for 1 hour). mTOR reactions are initiated by adding 0.1 mM ATP and 10 mM MgCl2 in the presence of various concentrations of KU-0063794 and GST-Akt1 (0.5 μg) or GST-S6K1 (0.5 μg). Reaction are carried out for 30 minutes at 30 °C on a vibrating platform and stopped by addition of SDS sample buffer. Reaction mixtures are then filtered through a 0.22-μm-poresize Spin-X filter and samples are subjected to electrophoresis and immunoblot analysis with the indicated antibodies.
|体外||DMSO||16 mg/mL (34.36 mM)|
30% PEG400+0.5% Tween80+5% propylene glycol
質量 (g) = 濃度 (mol/L) x 体積 (L) x 分子量 (g/mol)
開始濃度 x 開始体積 = 最終濃度 x 最終体積
この方程式は、一般に略語を使われます：C1V1 = C2V2 ( 入力 出力 )
チップス: 化学式は大文字と小文字の区別ができます。C10H16N2O2 c10h16n2o2